Poly-beta-hydroxyalkanoates (PHA's) are a heterogeneous family of biodegradable aliphatic polyesters Which include, for example, derived polymers such as poly-beta-hydroxybutyrate (PHB) and poly-beta-hydroxyvalerate (PHV).
PHB is an energy storage material produced by a variety of bacteria in response to environmental stress. Lemoigne discovered the presence of PHB in Bacillus in 1926 and it has since been identified in many different bacterial genera, including Azotobacter beijerinckia, Alcaligenes, Pseudomonas, Rhizobium, and Rhodospirillum. PHB is a homopolymer of D-(-)-3-hydroxybutyrate and has physical properties comparable to polypropylene. The biodegradability of PHB makes it especially suitable for a wide variety of purposes. PHB has also been used as a source of chiral centers for the organic synthesis of certain antibiotics, and has been utilized in drug delivery and bone replacement applications.
The biosynthesis of PHB has been studied extensively in A. eutrophus and Azotobacter beijerinckii. FIG. 1 outlines a three step biosynthetic pathway for PHB found in many prokaryotic organisms. Beta-ketothiolase first catalyzes the reversible condensation of two acetyl coenzyme A (CoA) molecules to acetoacetyl-CoA. The acetoacetyl-CoA is reduced by acetoacetyl-CoA reductase to D-(-)-3-hydroxybutyryl-CoA. Enzyme action of the acetoacetyl-CoA reductase is dependent on NADPH. PHB synthetase polymerizes the D-(-)-3-hydroxybutyryl-CoA to PHB.
PHB accumulates when growth of a bacteria culture is restricted by a nutrient other than a carbon source. For example, oxygen deprivation, nitrogen deprivation, sulfate limitation and magnesium limitation have all been used as limitations on environmental conditions. Under such environmental conditions, the PHB content in bacteria cells can increase to as much as 80% of the dry weight. When the limiting conditions are relaxed, PHB quantities decrease to preinduction levels. Induction studies have revealed that both beta-ketothiolase and acetoacetyl CoA reductase enzymatic activities increase markedly in response to PHB-stimulating limitation conditions.
It is now widely known that PHB can be accumulated in natural bacteria genera (for example, Alcaligenes eutrophus) using a wide variety of biotechnological processes and extraction methods. In addition, it has recently been known that PHA copolymers of PHB and PHV can be produced using various natural bacterial genera. One example of such copolymers is the subject of U.S. Pat. No. 4,876,331 to Doi and assigned to Mitsubishi Kasei Corporation. Another example of such copolymers is commercially available under the Biopol R trademark from Imperial Chemical Industries.
While PHA's can be produced in natural bacterial genera, these bacteria are less manipulatable and not as well characterized as E. coli. In the field of genetic engineering, a relatively large body of knowledge exists for E. coli. E. coli have been utilized as host cells for producing a wide variety of products including Human Growth Hormone, insulin and interferon.
The Slater et al., J. Biol. 170:4431 (October 1988) and the patent applications filed Jun. 7, 1989, Ser. No. 07/528,549, filed May 25, 1990 and Ser. No. 07/705,806, filed May 24, 1991, all of which are expressly incorporated herein by reference, describe the cloning of the PHB biosynthetic pathway from A. eutrophus into E. coli. The cloning of the PHB biosynthetic pathway into E. coli has also been later described by Schubert et al., J. Bacter. 170:5837 (December 1988); Peoples, et al., J. Biol. Chem., 264:15298 (Sept. 1989a) and Peoples et al., J. Biol. chem., 264:15293 (Sept. 1989b). There are several decided advantages of producing PHB by transformed E. coli over the production of PHB in A. eutrophus. First, the reproductive potential of E. coli is extremely high so that in a fermentation situation, it is possible to grow E. coli cells to a high concentration of biomass more quickly. Second, the accumulation of PHB in E. coli occurs to substantially higher levels than it does in A. eutrophus. The accumulation of PHB in E. coli (wt. of PHB/dry cell weight) as high as 95% has been obtained using the invention disclosed in the aboveidentified parent applications while highest accumulation of PHB in A. eutrophus is only around 80%. Third, since the PHB biosynthetic pathway is cloned on a plasmid, it is possible to make mutants that are even more efficient at PHB production than those disclosed in the above-identified patent applications. Fourth, E. coli enjoys the advantage of being able to use various different carbon sources. A. eutrophus does not possess this property. Lastly, E. coli is a well studied organism and there are a myriad of experimental techniques that can be used advantageously in other steps of PHB production. Thus, the prohibitive costs associated with A. eutrophus-produced PHB can be avoided such that it is commercially feasible to produce PHB on a large commercial scale.
The cloning of the PHB biosynthetic pathway found in A. eutrophus H16 into E. coli and expressing that pathway by the production of PHB in the cloned E. coli was disclosed in the parent Ser. No. 07/362,514 patent application. An A. eutrophus H16 library was constructed using cosmid pVK102. Cosmid clones that encoded the PHB biosynthetic pathway were sought by assaying for betaketothiolase. Six enzyme positive clones were identified and three of these clones manifested acetoacetyl CoA reductase activity and accumulated PHB. PHB was produced in the cosmid clones at approximately 50% of the level found A. eutrophus. One cosmid clone was subjected to subcloning experiments, and the PHB biosynthetic pathway was isolated on a 5.5 kilobase (kb) KpnI-EcoRI fragment, plasmid pSB20. This fragment can direct the synthesis of PHB in E. coli to levels approaching 50-55% of the bacterial cell dry weight.
A strain of E. coli, i.e., HMS174, has been transformed by a vector containing a plasmid (p4A) with the PHB biosynthetic pathway and approximately four hundred extra bases on both the upstream and downstream sides of the pathway. The HMS174 strain of E. coli contains a lactose utilization system and is recombination deficient so that a plasmid containing lactose genetic regions will not recombine and make the construct unstable. The strain of transformed E. coli can be grown in minimal media containing whey and has an average yield of PHB of approximately 85% (PHB dry weight/total cell dry weight).
However, until the present invention, it has been not possible to use the PHB transformed E. coli technology to produce PHA copolymers comprising PHB and other polymers. PHA's are generally defined by the carbon number of their backbone. For instance, the PHA that has a C4 backbone is poly-beta-hydroxybutyrate, whereas the PHA that has a C5 backbone is poly-beta-hydroxyvalerate. The size of the backbone has been shown to vary with the bacterial species and the carbon source. That is, certain species have only been shown to produce PHB, others produce PHB and PHV, still others produce PHA's such as poly-beta-hydroxydodecanoate. It is now understood that in order to produce PHA's having monomeric constituents higher than C4, there must be a carbon source in addition to/other than glucose. Thus, Alcaligenes eutrophus produces PHB when grown on glucose or gluconate, but produces PHB-co-V if valerate is added to the culture medium. Likewise, in order to induce Pseudomonas fluorescens to produce poly-beta-hydroxyoctanoate, the bacterium must be fed octane.
The importance of each PHA copolymer is that the plastic properties of each polymer vary considerably with the size of the carbon backbone of the monomer unit. PHB is considered to be a "brittle" plastic, whereas PHV is a more flexible plastic. In the same manner, poly-hydroxy-octanoate is a very flexible "rubbery" plastic.
This difference in plastic property allows PHB-co-V to be used in films, whereas PHB is not suited to plastic film applications, but more to molded products. Furthermore, the composition of the PHAs produced by bacteria can be varied by altering the culture conditions. In the case of A. eutrophus, valerate levels in the growing culture are altered to produce a PHB-co-V polymer that contains valerate monomer units of anywhere between 1% to 30% of the polymer. The flexibility of the copolymer increases as the percentage of valerate increases.